Melatonin promotes proliferation and differentiation of neural stemcells subjected to hypoxia in vitro

Introduction

Neural stem cells (NSCs) have the capacity for self-renewaland generation of new neurons and glial supporting cells.

They are present not only in the fetal brain but also in thenewborn and adult special brain areas in predictableproportions [1]. Furthermore, the proliferation and differ-

entiation activities of the NSCs do not remain static; ratherthey are dynamically regulated by various humoral andadhesive factors under physiological and pathophysiolog-ical conditions [2, 3]. Thus, the identification of different

extrinsic factors regulating NSCs activity may contribute toa further understanding of the neural ontogeny as well astoward the development of new therapeutic strategies

against neural injury.Melatonin (N-acetyl-5-methoxytryptamine) is a small

neurohormone that is highly soluble in both lipid and

water. Circulating melatonin is synthesized in the pinealgland as well as in peripheral tissues and secreted at high

levels in a circadian manner [4]. It can enter the centralnervous system (CNS) not only by readily crossing the

blood–brain barrier (BBB), but also via the pineal recess,and in damaged brain, directly from the circulation becauseof leaky BBB. Melatonin has a variety of important

physiological functions, including regulation of circadianrhythms, as well as visual, reproductive, cerebrovascular,neuroendocrine, and neuroimmunological actions [5, 6]. In

addition, melatonin exerts a neuroprotective effect in manypathological conditions of the CNS, such as Parkinson�sdisease, Alzheimer�s disease, and ischemic brain injury [7–11]. Moreover, its antioxidant function has attracted much

attention [12–14]. Recently, it has been reported thatmelatonin influences cell growth and differentiation of theNSCs [15–17]. However, its precise roles and the signaling

pathway involved in the process, especially under alteredconditions have remained unknown.This study investigated the effect and molecular mecha-

nism of melatonin on mouse NSCs under hypoxic condition

Abstract: Melatonin, an endogenously produced neurohormone secreted by

the pineal gland, has a variety of physiological functions and neuroprotective

effects. It can modulate the functions of neural stem cells (NSCs) including

proliferation and differentiation in embryonic brain tissue but its effect and

mechanism on the stem cells in hypoxia remains to be explored. Here, we

show that melatonin stimulates proliferation of NSCs during hypoxia.

Additionally, it also promoted the differentiation of NSCs into neurons.

However, it did not appear to exert an obvious effect on the differentiation of

astrocytes. The present results have further shown that the promotional

effect of NSCs proliferation by melatonin involved the MT1 receptor and

increased phosphorylation of ERK1/2. The effect of melatonin on

differentiation of NSCs is linked to altered expression of differentiation-

related genes. In the light of these findings, it is suggested that melatonin may

be beneficial as a supplement for treatment of neonatal hypoxic–ischemic

brain injury for promoting the proliferation and differentiation of NSCs.

Jie Fu1*, Shi-Dou Zhao1*,Hui-Juan Liu2, Qiu-Huan Yuan1,Shang-Ming Liu1, Yan-MinZhang1, Eng-Ang Ling3 andAi-Jun Hao1

1Key Laboratory of the Ministry of Education

for Experimental Teratology, Department of

Histology and Embryology, Shandong

University School of Medicine, Jinan,

Shandong, China; 2Bio-X Center, Key

Laboratory for the Genetics of Developmental

and Neuropsychiatric Disorders, Ministry of

Education, Shanghai Jiao Tong University,

Shanghai, China; 3Department of Anatomy,

Yong Loo Lin School of Medicine, National

University of Singapore, Singapore, Republic

of Singapore

Key words: differentiation, hypoxia,

melatonin, neural stem cell, proliferation

Address reprint requests to Ai-Jun Hao, Key

Laboratory of the Ministry of Education for

Experimental Teratology, Department of His-

tology and Embryology, Shandong University

School of Medicine, 44#, Wenhua Xi Road,

Jinan, Shandong 250012, China.

E-mail: aijunhao@sdu.edu.cn

*These two authors contributed equally to the

study.

Received November 30, 2010;

Accepted January 20, 2011.

J. Pineal Res. 2011; 51:104–112Doi:10.1111/j.1600-079X.2011.00867.x

� 2011 John Wiley & Sons A/S

Journal of Pineal Research

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in vitro. The effects and mechanism of melatonin on theproliferation, apoptosis and differentiation of NSCs wereevaluated. We determined if melatonin could promote

NSCs growth especially in hypoxia and, if so, the possibilityof its being used as a potential therapeutic agent for thetreatment of neonatal hypoxic–ischemic brain damage.

Materials and methods

Chemicals

The reagents and chemicals used in this study werepurchased from the following sources: melatonin and

poly-Lornithine hydrobromide from Sigma-Aldrich (StLouis, MO, USA); melatonin receptor antagonist (luzin-dole) from Tocris Bioscience (Ellisville, MO, USA).

Cell culture

Neural stem cells were prepared from the cerebral cortex ofKun Ming mouse (KM strain) at embryonic day 12.5(E12.5) as we described previously [18]. Briefly, the single

cells were prepared from telencephalon and seeded at2 · 105 cells/mL in DMEM/F12 (1:1) medium supple-mented with 20 ng/mL basic fibroblast growth factor(bFGF; R&D, Minneapolis, MN, USA), 2% B27 (Gibco,

Gaithersburg, MD, USA), plus 100 U/mL penicillin and100 lg/mL streptomycin. The cultures were incubated at37�C in a humidified atmosphere of 5% CO2 and 95% air.

After 3 days of culture, the cells proliferated and formedprimary neurospheres (about 80–100 lm in diameter).After 5–7 days of culture, the primary neurospheres com-

posed of NSCs were harvested by centrifugation, dissoci-ated using trypsin and EDTA (Sigma-Aldrich) into singlecells. The single cells were re-plated in serum-free mediumand cultured for 3–5 days (passage 1 neurospheres). All

experimental procedures were carried out using NSCsdissociated from passage 1 neurospheres.

To examine the proliferation of NSCs, the dissociated

NSCs were cultured for 3 days in proliferation mediumwith or without melatonin. For differentiation of NSCs,neurospheres were transferred into differentiation medium

containing 2% fetal bovine serum (FBS) without thegrowth factors and cultured for 3–9 days.

Hypoxia treatment of NSCs

Neural stem cells were placed in a modular chamber withhypoxic condition of humidified 95% N2 and 5% CO2

(BUGBOX; Ruskinn Technology, Pencoed, Bridgend, UK)for 12 hr. Both gas mixtures were saturated with H2Ovapor at 37�C. After this, they were returned to normoxic

conditions (95% air and 5% CO2) [19, 20].

Cell viability assays

The indirect counting of viable cells was determined by thetetrazolium salt MTT [3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. NSCs were plated

into 96-well culture plates at an optimal density of5 · 104 cells/mL with 200 lL culture medium per well.

After 24 hr, the cells were treated with different concentra-tions of melatonin. At 24 or 48 hr after melatonin treat-ment, 20 lL medium containing 5 mg/mL MTT was added

to each well and incubated at 37�C for 4 hr. The mediumwas gently aspirated, following which 200 lL DMSO wasadded to each well to solubilize the formazan crystals. Theoptical density of each sample was then measured in a

multi-well spectrophotometer at 490 nm. Four independentexperiments were conducted.Along with the above, hypoxia-treated NSCs which were

pretreated with 100 nm melatonin were plated into 96-wellculture plates at an optimal density of 5 · 104 cells/mL with200 lL culture medium per well. They were treated with

100 nm melatonin for 1–5 days, and then tested with MTTassay at different time points, respectively.

Proliferation index by bromodeoxyuridine labelingand immunostaining

Cell proliferation was evaluated by bromodeoxyuridine

(BrdU) incorporation assay. Hypoxia pretreated NSCs weretransferred into 12-well plates with poly-d-lysine coatedcoverslips, and incubated with BrdU (10 lmol/L; Sigma-

Aldrich) for 2 hr. The cultures were fixed in 4% parafor-maldehyde for 20 min, treated with 2 mol/L HCl at 37�C for30 min, blocked with 10% normal goat serum for 30 min,

and incubated with anti-BrdU monoclonal antibody(1:1000; Sigma-Aldrich) overnight at 4�C. Goat anti-mouseIgG coupled to TRITC (1:200; Chemicon, Temecula, CA,USA) was used as the secondary antibody. BrdU-positive

cells expressing nestin were detected by double immuno-staining with the respective specific antibodies.

Immunocytochemistry

Neural stem cells were plated on coverslips (5 · 104 cells per

well) coated with poly-d-lysine. After incubation, themedium was removed and the cells were washed twice withphosphate-buffered saline (PBS). The cells were then fixed in4% paraformaldehyde in PBS for 30 min at room temper-

ature and rinsed with PBS three times for 5 min andpermeabilized with 0.1% Triton X-100 for 20 min at roomtemperature. Subsequently, cells were incubated with

primary antibodies: anti-nestin (1:100, rabbit polyclonal;Abcam, Cambridge, MA, USA), anti-microtubule-associ-ated protein 2 (MAP2, 1:200, mouse monoclonal; Milli-

pore), anti-glial fibrillary acidic protein (GFAP, 1:500,mouse monoclonal; Millipore, Billerica, MA, USA).TRITC-conjugated goat anti-mouse IgG (Millipore) and

FITC-conjugated goat anti-rabbit IgG (Sigma-Aldrich)were used as secondary antibodies. Cell nuclei were count-erstained with 4¢, 6¢-diamidino-2-phenylindole (DAPI; Vec-tor Laboratories, Burlingame, CA, USA). Coverslips were

mounted in 90% glycerol/PBS and analyzed using afluorescence microscope (IX71; Olympus, Tokyo, Japan).

Western blot analysis

This was carried out following standard methods. Briefly,

NSCs harvested were washed with ice-cold PBS and lysedin cold lysis buffer containing 10 mm Tris HCl, pH 8.0,

Effects of melatonin on neural stem cells in hypoxia

105

240 mm NaCl, 5 mm EDTA, 1 mm dithiothreitol (DTT),0.1 mm PMSF, 1% Triton X-100, 1 mm sodium vanadate,and 1 g/mL of leupeptin, pepstatin, aprotinin by incubation

at 4�C for 20 min and centrifuged at 14,000 · g for 20 min.Protein concentration of the supernatants of cell extractwas determined using a BCA protein assay kit (PierceBiotechnology Inc., Rockford, IL, USA). Equal amounts of

proteins were loaded on 10% SDS-polyacrylamide gel.After electrophoresis, the proteins were transferred toPVDF membranes, and the blots were subsequently probed

with the following antibodies directed against: GFAP (N-19) (1:1000; Millipore), MAP2 (1:1000; Millipore), ERK1/2and p-ERK1/2 (1:1000; Cell Signaling Technology, Beverly,

MA, USA), MT1 (1:1500; rabbit polyclonal, Santa CruzBiotechnology, Santa Cruz, CA, USA), caspase-3 andcleaved caspase-3 (1:2000; Cell Signaling Technology),Bcl-2 (1:1500; Santa Cruz), and Bax (1:2000; Santa Cruz).

For detection, horseradish peroxidase-conjugated second-ary antibodies (1:5000; Sigma-Aldrich) were used followedby enhanced chemiluminescence development (Millipore).

Normalization of the results was done by running parallelWestern blots using b-actin as control. The optical densitywas quantified using an AlphaEase FC Version 4 analysis

software (Alphalmager HP; Alpha Innotech, San Leandro,CA, USA).

RNA extraction and Semiquantitative RT-PCR

Total RNA was extracted from mouse NSCs cultured with1 · 106 cells using the TRIZOL (Invitrogen, Carlsbad, CA,

USA) method. The amount of RNA obtained was deter-mined by spectrophotometric measurements at 260 nm.The sample was incubated for 10 min at 70�C followed by

addition of 4 lL of 5· transcription buffer, 2 lL of 0.1 m

DTT, 1 lL of 10 mm dNTPs, and 200 U of Superscript II(Invitrogen). The final volume of the reaction was 20 lL.The mixture was incubated for 60 min at 42�C. For PCRamplification, different amounts of the synthesized cDNA(diluted 1:10 in water) were analyzed to evaluate thelinearity of the reaction. Polymerase chain reaction was

then carried out in a solution containing 1.5 mm MgCl2,200 lm of each nucleotide in PCR buffer, 10 pmol of theprimers and 0.25 U platinum Taq DNA polymerase (Invi-

trogen). Primers for detecting genes are listed in Table 1.The PCR products separated on 1.5% agarose/TAE gelswere visualized by staining with ethidium bromide and

semiquantified using an AlphaEase FC Version 4 analysissoftware (Alphalmager HP; Alpha Innotech). The densito-metric analysis of the data was normalized to the b-actin.

Statistical analysis

Results are presented as mean ± S.D., each from three

separate experiments. Statistical difference was analyzed byone-way ANOVA followed by Dunnett�s test. P-value<0.05 was considered to be significant.

Results

The effects of various concentrations of melatonin on NSCsviability were first assessed. NSCs were incubated in the

growth medium in the presence of increasing concentra-

tions of melatonin (0, 1 nm, 10 nm, 100 nm, 1 lm, 10 lm,100 lm). MTT results showed that melatonin treatment for24 hr obviously improved the cell viability of NSCs in a

dose-dependent manner (10 nm, 100 nm, 1 lm melatoninwas 1.34 ± 0.13, 1.53 ± 0.11, 1.38 ± 0.12, respectively) incomparison with cells not treated with melatonin (Fig. 1).Furthermore, 100 nm melatonin yielded the optimal effect

on cell viability (1.53 ± 0.11 and 1.63 ± 0.16 at 24 and48 hr, respectively, P < 0.05).We then examined the effect of melatonin on the

proliferation of hypoxic NSCs in vitro. The cell viabilitywas first determined at 1–5 days after hypoxia and theresult showed that hypoxia decreased the cell viability

(P < 0.05). Melatonin treatment promoted the cell viabil-ity both in normal and hypoxic conditions at all timepoints, notably at 3 days after hypoxia (Fig. 2A). At 3 days

after hypoxia, the ratio of BrdU/nestin positive cells againsttotal nestin-positive NSCs (>95% of all cells) decreasedfrom 13 ± 1.2% to 9 ± 1.0% (P < 0.05). However, aftermelatonin treatment, the frequency of BrdU/nestin positive

Table 1. Sequences and PCR product sizes for each pair of primers

Genes Primer sequence (5¢–3¢) size (bp)

b-actin F: AGA TGT GGA TCA GCA AGC AG 104R: GCG CAA GTT AGG TTT TGT CA

Neurog1 F: CGA TCC CCT TTT CTC CTT TC 239R: TGC AGC AAC CTA ACA AGT GG

Mash1 F: AAG TCA GCG GCC AAGCAG GTC AAG

245

R: CGC AGC GTC TCC ACC TTGCTC ATC T

NeuroD2 F: GGC CGA AGA AAC GCA AGA TG 265R: GCA CAG AGT CTG CAC GTA GG

Hes1 F: CGA GCG TGT TGG GGA AGT A 101R: AGT GCG CAC CTC GGT GTT A

Hes5 F: GAT GCT CAG TCC CAA GGA GA 336R: CGC TGG AAG TGG TAA AGC AG

Id1 F: GAT CAT GAA GGT CGC CAG TAG 218R: GCT CCT TGA GGC GTG AGT A

Id2 F: CTC CAA GCT CAA GGA ACT GG 207R: ATG CTG ATG TCC GTG TTC AG

MT1 F: AGT GTC ATT GGC TCG GTA T 364R: GCT TCA GTT TGG GTT TGC T

Fig. 1. Dose-dependent effect of melatonin on cell viability ofneural stem cells (NSCs). NSCs were seeded on 12-well plates at adensity of 2 · 104 cells per well in growth medium with 0, 1 nm,10 nm, 100 nm, 1 lm, 10 lm, 100 lm melatonin for 24 and 48 hr.Cell viability was determined by MTT assay. Data (mean ± s.d. ofthree separate experiments) are expressed as fold over control value(no melatonin). *P < 0.05 as compared to the control.

Fu et al.

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cells was significantly increased compared with the un-treated cells (15 ± 1.3% versus 9 ± 1.0%, P < 0.05)(Fig. 2B).

To determine whether melatonin could affect the celldeath of NSCs in hypoxia, NSCs were stained with Hoechst

at 3 days after hypoxia. NSCs showing pyknotic nuclei(Fig. 3A, white arrows) indicative of cell death wereenumerated. The rate of cell death in hypoxia

(29 ± 1.6%) was significantly higher than that in thecontrol (7 ± 1.6%) (P < 0.05). In hypoxic cells treatedwith melatonin, the frequency of pyknotic nuclei decreasedfrom 29 ± 1.6% to 15 ± 1.3% (P < 0.05) (Fig. 3B).

For cell differentiation analysis, NSCs were exposed tohypoxic condition for 12 hr. The cells were then differen-tiated in differentiation medium (containing 2% FBS) in

normal condition or treated with melatonin to determinewhether it would influence the 2% FBS-induced NSCdifferentiation into MAP2-immunoreactive neurons and

GFAP-expressing astrocytes by immunocytochemistry at 3,5, 7, and 9 days after hypoxia. The percentage of neurons inrelation to the total cell number was decreased in hypoxia

group compared with the controls at all time points.Melatonin reversed this and increased the percentage ofMAP2-positive cells in all melatonin treated groups(P < 0.05), especially at 7 days differentiation (Fig. 4A)

as confirmed by Western blot analysis (Fig. 4B). Whencompared with the controls, hypoxia did not appear toaffect the differentiation of NSCs into astrocytes. In

Fig. 2. Effect of melatonin on the proliferation of neural stem cells(NSCs) in hypoxia. (A) Column graph showing the cell viability ofNSCs 1–5 days after hypoxia and matching controls (CT) with orwithout melatonin (MT) treatment. Values are mean ± S.D.Normoxic and hypoxic cells show comparable growth patterns.Time scale indicates days after hypoxia. *P < 0.05, #P < 0.01. (B)Immunofluorescence images showing increase in incidence of BrdU/nestin double-labeled cells bymelatonin treatment for 3 days both inthe CT and Hy groups. Note that hypoxia reduces the frequency ofBrdU/nestin double-labeled cells but is reversed by melatonintreatment. Scale bar represents: 50 lm (40·). *P < 0.05.

Fig. 3. Effect of melatonin on the cell death of NSCs in hypoxia.(A) Neural stem cells showing pyknotic nuclei (white arrows,Hoechst staining) as detected at 3 days of proliferation in control(CT) and hypoxia (Hy) with or without melatonin (MT) treatment.Scale bar represents: 50 lm (40·). (B) Column graph showing thepercentage of pyknotic nuclei. The difference among differentgroups is statistically significant. *P < 0.05.

Effects of melatonin on neural stem cells in hypoxia

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melatonin-treated groups, NSCs differentiation intoGFAP-positive astrocytes remained unchanged (Fig. 5A,B).

Next, we explored the possible molecular mechanismsthat might be linked to the proliferation of NSCs afterhypoxia by melatonin. Expression of phosphorylation ofthe main MAPK (ERK1/2) was found to change signifi-

cantly at different time points (Fig. 6A). Phosphorylationof ERK1/2 elicited by melatonin was evident as early as

Fig. 4. Melatonin promotes neuronal differentiation of neural stemcells (NSCs). (A) After hypoxic exposure for 12 hr, NSCs werecultured in the differentiation medium in the absence or presence ofmelatonin (100 nm) followed by immunostaining with anti-MAP2(red) at 3, 5, 7 and 9 days, respectively. Nuclei were counterstainedwith DAPI (blue). Note that percentage of MAP2-positive cells inhypoxia (Hy) + melatonin (MT) group at day 7 is significantlyhigher than that of the untreated hypoxia (Hy) group. Scale barrepresents: 50 lm (40·). *P < 0.05, #P < 0.01. (B) Western blotanalysis demonstrates MAP2 expression in NSCs in all groups atday 7. Note the significant increase in MAP2 levels in cells treatedwith MT either in the control or Hy group. *P < 0.05.

Fig. 5. Melatonin does not affect neural stem cells (NSCs) differ-entiation into astrocytes. (A) NSCs were subjected to hypoxia (Hy)for 12 hr. They were then cultured in the differentiation medium inthe absence or presence of melatonin (100 nm), followed byimmunostaining with anti-GFAP (green) at 3, 5, 7, and 9 days,respectively. Nuclei were counterstained with DAPI (blue). There isno noticeable difference in GFAP immunofluorescence betweengroups. Scale bar represents: 50 lm (40·). (B) Western blot anal-ysis demonstrates GFAP expression in NSCs at day 7. GFAP levelsin control (CT) + melatonin (MT) and Hy + MT groups are notsignificantly different from the untreated groups.

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15 min, peaking at 60 min and declined at the 75 min(Fig. 6A). Melatonin receptor-MT1 mRNA and proteinwere detected in NSCs by RT-PCR and Western blot,

respectively (Fig. 6B,C). To determine whether the melato-nin-induced increase in NSCs proliferation acts via themelatonin receptor, NSCs were pretreated with 10 lm

luzindole, a melatonin receptor antagonist, prior to the

addition of 100 nm melatonin. Phosphorylation of ERK1/2induced by melatonin was significantly reduced (Fig. 6D).BrdU incorporation assay showed that cell proliferation in

the presence of melatonin and luzindole was similar to thecontrol group (data not shown).

It has been reported that hypoxia induces apoptotic cell

death by activating caspase-3 [21]. We therefore examinedchanges in hypoxia-induced caspase-3 activity followingmelatonin treatment of NSCs for 3 days. Melatonin treat-ment was found to suppress the caspase-3 activity which

was increased in hypoxia (Fig. 7A). To clarify the mech-anism underlying the protective effect of melatonin,Western blot analysis was performed to assess the expres-

sion of Bcl-2 and Bax in all groups at 3 days after hypoxia.Treatment of melatonin was shown to induce Bcl-2expression and increase Bcl-2/Bax ratio (Fig. 7B). This

suggests that melatonin inhibits caspase-3 activity andinduces Bcl-2 expression, thereby inhibiting apoptotic celldeath in NSCs.

To determine the mechanism of melatonin by which itcan regulate differentiation of NSCs, we investigated somebHLH transcription factors which determine the neuronand astrocyte fates from NSCs. NSCs were cultured under

hypoxic condition for 12 hr. The mRNA expression levelsof Mash1, Neurog1, NeuroD2, Hes1, Hes5, Id1, and Id2 in

differentiated cells were analyzed at 7 days after differen-tiation. The mRNA levels of Mash1, Neurog1, andNeuroD2 were significantly increased in melatonin treat-

ment groups, while the mRNA levels of Hes1, Hes5, Id1,and Id2 had no significant differences in all groups (Fig. 8).

Discussion

Neonatal hypoxic–ischemic brain injury is usually causedby perinatal asphyxia and hypoxia is pivotal to the

pathogenesis. The hypoxic insult results in acute anddelayed neuronal cell death through the activation of acomplex series of events, which ultimately lead to severe

brain dysfunction [22, 23]. Although many molecularmechanisms have been identified in hypoxic–ischemicencephalopathy, the intervention strategies are still limited[23, 24]. In contrast to adult brain, the developing brain is

considered to have great potential for regeneration afterinjury. Although increased cell proliferation and neurogen-esis after neonatal hypoxic–ischemic injury have been

reported in animal models [25–27], the intrinsic ability ofendogenous NSCs for neural self-repair is rather limited[28, 29]. In this connection, a promising strategy would be

to supplement the damaged brain with exogenous neuro-trophic factors to fully activate the endogenous NSCs forproliferation and differentiation after hypoxia. Such a

strategy targeting at the NSCs may promote brain repairand compensate for the impaired brain functions.In the present study, we have used an in vitro model to

study the effect of melatonin on NSCs subjected to hypoxic

exposure. First, we found that melatonin increased the cellviability of NSCs in a concentration-dependent manner,

(A)

(B)

(C)

(D)Fig. 6. MAPK signal is involved in mel-atonin-induced proliferation of neuralstem cells (NSCs). (A) Western blot anal-ysis shows time-dependent effects of mel-atonin on activation of ERK1/2 in NSCsin hypoxia. After hypoxia, NSCs weretreated with melatonin (100 nm) for 0, 15,30, 60, and 75 min. Western blot analysisshows melatonin increases p-ERK1/2 lev-els significantly. (B and C) MT1 mRNAand protein are detected by RT-PCR(397 bp) and Western blot analysis(45 kDa), respectively. (D) Hypoxic NSCswere treated with melatonin (100 nm) for0, 30, and 60 min with or without pre-treatment of melatonin receptor antago-nist (luzindole, 10 lm) for 6 hr asindicated. Note significant reduction inp-ERK1/2 with luzindole treatment. Val-ues represent mean ± s.d. *P < 0.05; n,not significant.

Effects of melatonin on neural stem cells in hypoxia

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but this stimulatory effect declined at the highestconcentration of melatonin (100 lm). This is consistent

with results of two other similar studies [16, 17]. Underanoxic conditions, the results have shown that hypoxiaresults in decreased cell viability and proliferation of NSCs,

in agreement with previous data on human NSCs [20].Remarkably, melatonin pretreatment reverses this and canincrease cell viability and proliferation of NSCs. In addi-

tion, the increased growth of NSCs under melatonin

treatment appears to reflect reduced cell death, leading tothe enhancement of self-renewal capacity.

It has been reported that melatonin suppressed theproliferation of NSCs derived from the mouse embryostriatum [15] and had no effect on the proliferation of

hippocampal neural precursor cells of adult mice [30]. Thisis inconsistent with our present results as well as otherstudies [16, 17, 31] which demonstrated that melatonin

increased the proliferation of NSCs. The reason for the

(A) (B)

Fig. 7. Melatonin affects the survival of NSCs via Bcl-2. (A) Inhibition of caspase-3 activity by melatonin. Neural stem cells were subjectedto hypoxia, followed by Western blot analysis of caspase-3 fragmentation at 3 days, with or without melatonin treatment. Cleaved caspase-3levels in melatonin-treated group are significantly lower than that of the untreated group. *P < 0.05. (B) Western blot analysis showingexpression of Bcl-2 and Bax. The ratio of Bcl-2/Bax in melatonin treated group is significantly greater than that of the untreated group.*P < 0.05.

Fig. 8. Melatonin affects expression of some bHLH transcription factors. (A) RT-PCR shows the mRNA levels of proneuronal bHLHtranscription factors (Mash1, Neurog1, and NeuroD2) at 7 days in differentiated cells. The mRNA levels of Mash1, Neurog 1 (NG1), andNeuroD2 (ND2) are significantly increased by melatonin (MT or Hy + MT) when compared with the matching controls (CT or Hy).b-Actin was used as an internal control. Data were obtained from three independent measurements. Values represent mean ± S.D.*P < 0.05, #P < 0.01. (B) RT-PCR shows the mRNA levels of inhibitory bHLH transcription factors (Hes1, Hes5, Id1, and Id2) at 7 daysin differentiated cells. The mRNA levels of these transcription factors were not significantly changed by melatonin when compared with thematching controls (CT or Hypoxia). b-Actin was used as an internal control. Data were obtained from three independent measurements.Values represent mean ± s.d.

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discrepancy is unclear. It is suggested that the different timepoints and culture conditions of stem cells or melatoninconcentrations used may be possible contributing factors.

Previous studies have shown that ERK1/2 is activated byphosphorylation in response to melatonin in neuronal cellcultures [32]. The MAP kinase signaling pathway regulatesthe suppression of cell death, promotion of cell growth,

differentiation and survival [33–35]. To elucidate themechanism underlying the above findings, we examinedthe ERK1/2 activation induced by melatonin. Melatonin

induced the activation of ERK1/2. The detection of MT1mRNA and protein in NSCs supports that melatoninmodulates the function of NSCs via the membrane mela-

tonin receptor. The stimulatory effects of melatonin onNSCs proliferation and the activation of ERK1/2 via MT1were further supported with the use of luzindole whichdecreased p-ERK1/2 significantly.

Melatonin is a well-known free radical scavenger andantioxidant [36–39] and thus as an anti-apoptotic agent[40–42]. Recently, some reports examined the relationship

between melatonin, Bcl-2 and caspase-3 expression [40, 43,44]. Especially, it has been reported that the protectiveeffect of melatonin against injured cerebral neurons is

related to Bcl-2 and caspase-3 protein. In this study, it wasdemonstrated that the protective effect of melatonin againsthypoxia-mediated apoptosis in NSCs is associated with the

induction of Bcl-2 expression and caspase-3 activation.Melatonin treatment induced a significant increase in theratio of Bcl-2/Bax and inhibition of caspase-3 activationand the reduced incidence of apoptosis.

In the present study, hypoxia reduced differentiation ofNSCs into neurons in the differentiation medium. Melato-nin treatment, however, promoted the process. The precise

mechanisms by which neurogenesis and gliogenesis areregulated in the NSCs, however, remain to be elucidated. Ithas been reported that melatonin treatment in NSCs can

significantly increase histone H3 acetylation, which isassociated with chromatin remodeling and gene transcrip-tion [45]. In neurodevelopment, determination of neuron

and glia cell fates from NSCs during fetal and adult braindevelopment depends on some basic helix–loop–helix(bHLH) factors [46–48]. The bHLH factors such as Mash1,Neurog1, and NeuroD2 are involved in vertebrate neuro-

genesis. We show here that melatonin treatment inducedup-regulation of Mash1, Neurog1, and NeuroD2 in thedifferentiated NSCs compared with untreated cells in

hypoxia. It is possible that increased neurogenesis causedby melatonin supplementation is the consequence ofenhanced expression of these transcription factors.

During neurodevelopment, other bHLH factors such asHes1, Hes5, and Id1 can regulate the specification ofgliogenesis [49–51]. The present results suggest that glio-genesis is not affected by melatonin. This is because there is

no significant difference in the expression of Hes1, Hes5,Id1, and Id2 with or without melatonin treatment.

Taken together, our findings suggest that melatonin

treatment for NSCs in hypoxia is a powerful strategy thatcan increase cell proliferation, reduce cell death, andenhance neuronal differentiation without affecting the

astroglial differentiation. Because melatonin can easilyenter the CNS, this intrinsic modulator might be benefi-

cially used for stimulating endogenous NSCs. It is sug-gested that a better clarification of the sites and mechanismsunderlying the induction and modulation by melatonin may

lead to development of novel strategies for purposes ofexpansion and differentiation of endogenous NSCs inhypoxia injury disease.

Acknowledgements

This research was supported by National Basic Research

Program of China (973 Program), Grant Number:2007CB512001, 2011CB966201; National Natural ScienceFoundation of China, Grant Number: 30771142, 81071057;

Natural Science Foundation of Shandong Province, GrantNumber: Z2007C11, J200823 and ZR2010HQ022.

Author contributions

JF, SDZ and HJL: conception and design, performance ofexperiments, data analysis and interpretation, manuscript

writing; QHY, SML and YMZ: data analysis and inter-pretation; EAL: data analysis and interpretation, manu-script writing; AJH: conception and design, financial

support, data analysis and interpretation, manuscriptwriting and final approval of manuscript.

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